The staff of the Protein Purification Core (PPC) use a number of techniques for effective protein production. The PPC has access to a wide variety of tools for the expression of recombinant protein in Escherichia coli, including many types of plasmid expression vectors and specialized bacterial strains. There is a rather large collection of strains to choose from, with genetic defects that influence proteolytic activity, mRNA stability, membrane permeability, and intracellular redox potential. In addition, there are strains that overproduce protein disulfide isomerase, molecular chaperones, and redox enzymes for coexpression with target proteins. There is an equally large and diverse collection of bacterial plasmid vectors for recombinant protein expression. Many of these use the Gateway cloning technology (Life Technologies), making them quick and easy to use. The PPC staff has experience with all of the major regulatory systems (e.g., T7, tac, pBAD, trc, lambda PL) and various formats for the production of recombinant proteins (untagged or fused to MBP, GST, NusA, thioredoxin, His-tag, Arg-tag, FLAG-tag, biotin acceptor peptide, etc.) to make full use of these reagents. The PPC has recently added an insect cell protein production facility to compliment its bacterial production capability, using the Bac-to-Bac baculovirus expression system (Life Technologies). The facility is up and running and like most bacterial production utilizes Gateway cloning technology to maximize productivity. This year the PPC has expanded its insect cell production to include two additional technologies, the flashBAC baculovirus expression system (Oxford Expression Technologies) and the Drosophila expression system (Life Technologies). Although still in development, indications are that these systems may be better for secretory and membrane-bound protein production.The PPC personnel are experienced with all standard chromatography techniques required for protein purification. The core maintains a full array of supplies necessary for ion exchange, hydrophobic interaction, lectin, hydroxyapatite, dye, size exclusion, and affinity chromatography. Materials for chromatofocusing are also on hand. In addition to purification technology, the staff is very knowledgeable of methods required to characterize recombinant protein products. Among those used are gel electrophoresis and isoelectric focusing, mass spectroscopy, western analysis, N-terminal sequencing, dynamic light scattering and analytical ultracentrifugation, and circular dichroism spectroscopy. For structural studies, the PPC has in place standard operating procedures for the production of isotopically enriched proteins for heteronuclear Nuclear Magnetic Resonance experiments and selenomethionine-substituted proteins for crystallography. Methods have been established for bacteria that eliminate the need to change cell type by manipulating the medium formulation and induction parameters, and produce recombinant protein at levels equivalent to the wildtype expression. For those proteins that fail to crystallize, the core can perform limited proteolysis as a way to identify potential structural domains, providing the Macromolecular Crystallography Laboratory investigator additional avenues for structural studies. This method has been extensively used both analytically, and on a preparative scale to produce structural domains that can be purified using conventional chromatography. The core produces and maintains three different kinds of tobacco etch virus (TEV) protease that are used by the Macromolecular Crystallography Laboratory for in vitro cleavage of fusion proteins that contain an intervening protease recognition sequence. Available are an N-terminal tagged His7-TEV protease, an untagged TEV protease and a Maltose Binding Protein-TEV protease fusion protein. All contain a mutation that minimizes autoinactivation. Each has its advantage depending on the design of the protein purification scheme.Similarly, the core produces and maintains two types of tobacco vein mottling virus (TVMV) protease also used for in vitro cleavage of fusion proteins. These are available as an N-terminal His6-tagged TVMV protease and an untagged TVMV protease. The protease recognition site is different from TEV protease and allows the use of both recognition sequences in a single fusion protein. As an alternative to these potyvirus proteases, the PPC this year has purified the human rhinovirus 3C protease (i.e., PreScission protease) using a bacterial expression plasmid obtained from Arie Geerlof (Helmholtz Center Munich, Institute of Structural Biology, Neuherberg, Germany). This protease has good activity even at 4C and will be quite useful in cleaving fusion proteins produced in several commercial vectors such as the pGEX-P series, pTriEx-9 and pET-47b(+). In addition, the PPC has also purified the glycosidase endo-beta-N-acetylglucosaminidase H using a bacterial expression plasmid obtained from Daniel Leahy (Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland). This enzyme will be invaluable at removing asparagine-linked oligosaccharide side chains from proteins produced in our insect cell protein production facility, which often impede the crystallization process.For FY2012, as part of our research support for the Macromolecular Crystallography Laboratory, the PPC has completed 42 cloning projects and performed 65 protein purification. In addition 114 insect cell protein productions at the pilot and preparative levels were completed.